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Animal Model |
From the Division of Nephrology,* Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; and the Department of Pathology, University of Florida, Gainesville, Florida
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Abstract |
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Previous studies have used chemical inhibitors or inducers of HO-1 to evaluate the immunomodulatory functions of HO-1. However, both chemical inducers (such as hemin) and inhibitors (such as tin or zinc protoporphyrin) of HO-1 have effects beyond altering HO-1 enzyme activity per se.7 In addition to inducing HO-1, hemin induces other genes as well (eg, adhesion molecules), many of which influence important cellular processes.8 Hemin also exhibits pro-oxidant properties and recent studies have shown that it causes mitochondrial injury.9,10 The metalloporphyrin inhibitors of HO-1 affect other enzyme systems such as NO synthase and guanylate cyclase, in addition to a variety of nonspecific effects.11,12 For such reasons, the use of transgenic mice either genetically deficient or overexpressing HO-1 will provide further insight for a role of HO-1 in the mediating its immune effects.
The development of the HO-1 knockout mouse and the description of the patient with HO-1 deficiency have significantly advanced our knowledge pertaining to the role of HO-1 in disease pathophysiology.13 Using HO-1 knockout mice, we and others have demonstrated a functional role for HO-1 in several models of tissue injury.7,14,15 However, the general role of HO-1 in forming an immune response has not yet been investigated in these mice. The purpose of the present study was to examine the immune phenotype associated with HO-1 deficiency as well as the influence of HO-1 activity on the immune response following mitogen stimulation.
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Materials and Methods |
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Male HO-1–/– mice (8 to 12 weeks of age, C57BL/6xFVB) carrying a targeted deletion of a large portion of the HO-1 gene, were selected from offspring of heterozygous/homozygous mating by PCR using tail DNA as previously described.5,14 Age-matched wild-type (+/+) littermates were used as controls. The original breeding colony of the HO-1+/– mice was obtained from Dr. Susumu Tonegawa’s laboratory (Massachusetts Institute of Technology, Cambridge, MA) and was of a predominantly C57BL/6 background. The yield of homozygous mice from this colony was very low (average of about 1 to 2 homozygotes per 160 to 200 pups from heterozygote breeding pairs) as was reported in the original publication by Poss and Tonegawa.5 We backcrossed C57BL/6 HO-1+/- mice with wild-type FVB mice. The HO-1+/– offspring of these mice was backcrossed to wild-type FVB mice and these backcrosses were repeated four to five times, to derive a strain of HO-1+/– mice with a predominant FVB background. These mice were used in future matings, which resulted in an increased yield of HO-1–/– mice to about 1 in 20 pups. The study protocol was approved by the Institutional Animal Care and Use Committee at the University of Florida, Gainesville, FL.
Histology and Immunohistochemistry
Animals were sacrificed and lymphoid organs including the thymus, spleen, and lymph nodes collected. The tissue was fixed with either 10% formalin for hematoxylin and eosin staining (H&E) or in paraformaldehyde-lysine-periodate for immunohistochemistry. Ferric iron was detected in tissues using a Prussian blue stain. In brief, paraffin sections were deparaffinized and hydrated. Slides were heated in 2% potassium ferrocyanide/2% hydrochloric acid for 20 minutes at 60°C. Slides were washed in water and counterstained in nuclear-fast red for 5 minutes before mounting. The immunohistochemical staining for HO-1 was performed using a polyclonal rabbit anti-rat HO-1 antibody (1:300, catalog no. SPA-895, Stressgen Biotechnologies, Victoria, BC), and the binding was visualized with 3, 3'-diaminobenzidine substrate using an ABC-peroxidase kit (Rabbit Elite HRP kit, Vector Laboratories Inc., Burlingame, CA) with hematoxylin (DAKO Corp., Carpinteria, CA) counter stain. Spleen, thymus, and lymph nodes were stained using monoclonal rat antibodies against B cell (anti-mouse B-220, 1:50, catalog no. 550286, BD Biosciences Pharmingen, San Diego, CA), T cell (anti-human CD3, 1:500, catalog no. MCA1477, Serotec Inc., Raleigh, NC), and macrophage (anti-mouse CD11b, 1:200, catalog no. MCA74G, Serotec) antigens detected with a rat alkaline phosphatase kit (Rat AP kit, Vector Laboratories) containing biotinylated anti-rat secondary and ABC-AP conjugate. Blue chromogen with nuclear-fast red was used as a counterstain (Vector Laboratories).
Western Blot Analysis
For HO-1 and HO-2 immunoblots, spleens were washed twice with ice-cold PBS and lysed in a buffer containing a broad spectrum mixture of protease inhibitors consisting of 10 µg/ml aprotinin, 5 mmol/L EDTA, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 1 mmol/L phenylmethanesulfonyl fluoride, and Triton X-100. Protein concentration of lysates was assessed by the bicinchoninic acid assay (Pierce, Rockford, IL). Samples were separated in a 10% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The membranes were incubated for 1.5 hours with the anti-HO-1 antibody (1:500 dilution, catalog no. SPA-895, Stressgen) followed by incubation with peroxidase-conjugated goat anti-rabbit IgG antibody (1:10,000 dilution) for 1 hour. Labeled protein bands were examined by using a chemiluminescence method according to the manufacturer’srecommendation (Amersham, Piscataway, NJ). The membranes were then stripped and re-probed with anti-HO-2 (1:1000) (SPA-897, Stressgen) and anti-actin (1:1000) (Sigma, St. Louis, MO) antibodies.
Flow Cytometry
Splenocytes were isolated in a standard fashion and suspended cells were stained with monoclonal antibodies against B cell (B-220), T cell (CD3, CD4, and CD8), and macrophage (CD11b) antigens (BD Biosciences Pharmingen), coupled with various chromogens (phycoerythrin or FITC) and then fixed with BD Cytofix (BD Biosciences Pharmingen). Appropriate controls and gating were used to analyze the staining on a flow cytometer (FACS Calibur; BD Biosciences).
Splenocyte Stimulation Assays
Isolated splenocytes were cultured under respective stimulation conditions at 1 x 105
cells/well in 200 µl of RPMI 1640 medium with 10% FBS (Cambrex, East Rutherford, NJ) in 96-well round-bottom microculture plates (Fisher Scientific, Pittsburgh, PA). The supernatants were collected at 48 hours for cytokine analysis after stimulation with LPS (1 µg/ml), anti-CD3 and anti-CD28 antibodies (1 µg/ml), or medium alone (unstimulated). Cytokine measurements of supernatants were performed using a commercially available multiplexed kit (Beadlyte Mouse Multi-Cytokine Detection System, Upstate, Lake Placid, NY) and the Luminex100 LabMAP System (Luminex Corp., Austin, TX). Measurement of 10 cytokines included: IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), TNF-
, IFN-
, and GM-CSF. The assay was performed according to the manufacturer’s protocol.
Serum Immunoglobulin Measurement
Serum samples from animals were collected at the time of sacrifice and levels of IgA, IgM, IgE, IgG2a, and IgG3 assayed using Beadlyte Mouse Immunoglobulin Isotyping Kit (Upstate) and Luminex100 LabMAP System (Luminex Corp).
Statistical Analysis
Data are presented as the mean ± SEM. All results are derived from 3 to 7 animals per group. Two-tailed Student’s t-test were used for analyses comparing the different groups, with statistical significance considered if P < 0.05.
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Results |
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To assess the phenotypic characteristics of the newly derived genetic background in comparison to the original description of the murine HO-1 knockout,5
we performed a series of morphological studies. Consistent with previous studies,5,7
the mean body weights of the HO-1+/+ and HO-1–/– mice were not significantly different (26.7 ± 1.4 versus 25.4 ± 2.6 g, respectively, P = NS) in the age matched group of mice studied (8 to 12 weeks). The HO-1–/– mice showed significant splenomegaly in comparison to the HO-1+/+ littermates (220.6 ± 35.4 versus 68.3 ± 7.9 mg, respectively, P < 0.001), findings similar to the observations of Poss and Tonegawa.5
As shown in Figure 1A
, histological examination of the spleen from the HO-1–/– mice revealed abnormal architecture associated with significant fibrosis. The absence of HO-1 protein in the HO-1–/– mice was confirmed by immunohistochemistry (Figure 1A
, inset). While significant tissue iron deposition was noted in the kidneys and livers of HO-1–/– animals over 20 weeks of age (Figure 1B)
, no iron deposition was detectable by Prussian blue staining in the age group of animals used in our studies (data not shown). We also performed Western blot analysis for HO-1 and HO-2 proteins on spleens from the HO-1–/– and HO-1+/+ mice to evaluate for possible compensatory changes of HO-2 levels in the HO-1–/– mice. As shown in Figure 2
, despite absolute lack of HO-1, no increase of HO-2 protein was observed in spleens from HO-1–/– mice as compared to HO-1+/+ animals.
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. Immunohistological evaluation of the spleen from HO-1–/– mice revealed an abnormal distribution of CD3- and B220-positive cells within individual follicles (Figure 3C)
. CD3-positive cells were normally concentrated in the central region and B220 in the marginal zone of the follicle in HO-1+/+ mice, whereas this pattern was disturbed in HO-1–/– mice (Figure 3C)
. Since the number of CD11b-positive cells appeared to be increased in HO-1–/– spleens (Figure 3C)
, we performed a flow-cytometric analysis of various splenic cell populations. As demonstrated in Figure 3D
, no significant differences were observed in the numbers of CD3-, CD4-, CD8-, B220-, and CD11b-positive cells in the HO-1–/– compared to HO-1+/+ mice.
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To assess the immunoglobulin profile in the HO-1–/– and HO-1+/+ mice, we measured baseline serum levels of IgA, IgG2a, IgG3, IgE, and IgM. As shown in Figure 4
, IgM levels were significantly elevated (
3-fold) in the HO-1–/– compared to the HO-1+/+ mice, while no differences were noted for the other immunoglobulins tested.
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To assess whether there were any significant differences in immune responses between the HO-1–/– and HO-1+/+ phenotypes, we performed splenocyte stimulation studies followed by cytokine measurements in supernatants. The stimulation was performed either with LPS (for primarily monocyte/macrophage stimulation) or with anti-CD3/anti-CD-28 antibodies (for T-cell stimulation). The levels of IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), TNF-, IFN-, and GM-CSF were measured. Under baseline, unstimulated conditions, the levels of all cytokines measured were undetectable in both HO-1–/– and HO-1+/+ splenocytes. As shown in Figure 5A
(left panel), LPS stimulation resulted in a significant increase of IL-1, IL-6, IL-10, IFN-, and TNF- in HO-1–/– compared to the HO-1+/+ splenocytes. The most significant changes were observed with IL-1, IL-6, and TNF- (Figure 5A
, right panel) where the fold increase was 27, 37, and 19, respectively, in HO-1–/– over HO-1+/+ mice.
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, left panel), splenocytes from the HO-1–/– mice showed a significantly higher secretion of all cytokines, except IL-1 and IL-5. In these studies, the fold increase was disproportionately higher for GM-CSF (239-fold) and IL-6 (47-fold) in the HO-1–/– over HO-1+/+ mice (Figure 5B
, right panel). Collectively, these data suggest that HO-1 deficiency is associated with an exaggerated monocyte/macrophage and T cell activation yielding a predominantly proinflammatory state. |
Discussion |
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It is becoming more apparent that HO-1 activity not only provides protection in oxidant injury, but is also involved in direct modulation of the immune response.2,26
The mouse model of HO-1 deficiency provides the unique opportunity to study this involvement. In short, our studies provide evidence indicating a significant difference in the immune phenotype between HO-1–/– and HO-1+/+ mice. The studies indicate that a deficiency of HO-1 may predispose to generally exaggerated inflammatory responses, suggesting that its activity is necessary for timely resolution of early inflammation. Moreover, our data showing the predominance of Th1-type cytokines (eg, IL-1, IFN-, TNF-, IL-6) following splenocyte stimulation suggest that HO-1 activity is also important in more downstream stages of the immune response (eg, modulation of lymphocyte activation). The latter activity is of particular interest given the recent findings that HO-1 is constitutively expressed in a subpopulation of T regulatory cells (CD4+CD25+), and that the HO-1 level increases even further following T cell stimulation.27
The morphological data presented in this report demonstrate that the altered genetic background of our HO-1 knockout colony (C57Bl/6 x FVB) yields a very similar phenotype to the one originally reported. Specifically, the initial description of HO-1 knockout mice by Poss and Tonegawa5 has involved animals on a C57Bl/6 background. They have noted that animals lacking HO-1 developed progressive inflammatory disease characterized by splenomegaly, lymphadenopathy, leukocytosis, and hepatic and renal inflammation. The animals display increased iron accumulation5 and are more sensitive to oxidative stress.28 Since a full spectrum of the phenotypic changes associated with HO-1 deficiency is apparent in animals older than 20 to 24 weeks, we have restricted our functional studies to younger mice aged 8 to 12 weeks. At this point, the HO-1-deficient mice, although exhibiting marked splenomegaly, do not have any significant differences in their body weights, tissue iron deposition, or in relative numbers of various spleen cell subtypes minimizing thereby a potential influence of age-related phenomena.
Although it has been known that the phagocytosis of erythrocytes induces HO activity in rodent and porcine macrophages,29,30 Clerget and Polla31 have first proposed that the up-regulation of HO-1 might have an immunomodulatory function in addition to its obvious role in heme metabolism. The role of HO-1 in inflammation has been further explored by Willis et al32 in a model of pleural inflammation. In that system, prior induction of HO-1 (with hemin) results in a significant decrease in inflammatory cell infiltration and exudates whereas inhibition of HO-1 (by tin protoporphyrin) significantly worsens inflammatory exudates, suggesting that HO-1 activity modulates the inflammatory response. In the context of inflammation, our findings of a dramatic increase in pro-inflammatory cytokines following macrophage stimulation in the HO-1 knockout provide additional evidence for the importance of HO-1 in macrophage function. These studies correlate with the recent report that HO-1 knockout mice show a significant up-regulation of monocyte chemoattractant protein (MCP-1) in the kidney following repeated exposure to heme as well as higher serum levels of MCP-1 at baseline.33,34
The exact mechanisms involved in the broad range of anti-inflammatory and cytoprotective abilities of the HO-1 system have not been fully elucidated. One or more of the HO-1 reaction products have been evaluated as possible factors. For example, CO has been shown to exert significant anti-inflammatory and anti-apoptotic effects in several models of inflammatory tissue injury. CO has been shown to decrease IL-6 production from LPS-stimulated macrophages by interfering with AP-1 binding to the IL-6 promoter via a JNK-dependent pathway.35 Our results demonstrate a significantly higher IL-6 secretion following LPS stimulation from the HO-1–/– splenocytes. One could speculate that exposure of HO-1–/– splenocytes to CO should potentially attenuate the increase in IL-6 release. Otterbein et al22 have demonstrated that CO also exerts anti-inflammatory effects, in part, by increasing macrophage IL-10 production. Inoue et al36 have corroborated these findings by providing evidence that the overexpression of exogenous HO-1 in macrophages leads to a significant increase in macrophage-derived IL-10 levels. In contrast, Lee and Chau37 have reported that IL-10 induces HO-1 in macrophages and is responsible for its anti-inflammatory properties.
It should be noted that biliverdin and/or bilirubin are also capable of blocking key events in inflammation. Up-regulation of HO-1 activity has been shown to interfere with leukocyte adhesion to vascular endothelium via changes in expression of various adhesion molecules,38-40 a phenomenon that has been attributed to biliverdin and/or bilirubin, rather than CO.38,39 Elevated HO-1 levels correlate with a reduction in monocyte chemotaxis in response to oxidized LDL, an effect that is reversed by biliverdin and bilirubin.41 Our findings in HO-1 knockout mice provide a basis for the evaluation of the individual components of the HO-1 system in mediating its anti-inflammatory effects.
In our studies, we have observed significant differences in the serum immunoglobulin profile between the HO-1–/– and HO-1+/+ mice. The results show that HO-1 deficiency is associated with significantly higher baseline levels of IgM. This finding suggests a possible abnormality in B cell activation with impaired immunoglobulin isotype switching and is consistent with the work of Coito et al,42 who has reported that HO-1 induction following blockade of selectin-P-selectin glycoprotein ligand-1 interaction is associated with a decrease in IgM levels in a cardiac allograft model. A direct causal effect of increased HO-1 activity on B cell function has, however, not been established and will require further investigation.
The induction of HO-1 clearly plays an important role in the immune processes associated with transplant rejection where it positively correlates with transplant survival. Using a xenotransplant model, Soares et al15
has reported that cardiac transplants from HO-1–/– mice into rats have been rejected within 3 days whereas those from wild-type or heterozygous donors have survived for up to 60 days. Further evidence for the graft-protective properties of HO-1 have been provided in heart,42-45
liver,46,47
kidney,48
thyroid,49
and pancreatic islet50
allografts as well. Ke and associates46
have shown a correlation between high and low HO-1 levels in liver allografts and changes in cytokine profile within the infiltrated allograft toward the predominance of Th2-like molecules (IL-4 and IL-10) versus Th1-associated ones (IFN- and IL-2). Here also, high levels of HO-1 have been associated with increased allograft survival. These authors have also demonstrated a similar cytokine profile change in liver allograft recipients treated with a CO donor, methylene chloride, suggesting that CO might be responsible for the immunomodulatory properties of HO-1.47
In a model of kidney transplantation, Tullius et al48
have observed that the induction of HO-1 results in a significant reduction of graft infiltration with monocytes/macrophages and CD8-positive T cells. This has also been associated with a decrease in graft tissue TNF- mRNA levels as well as a reduction of IFN- mRNA. Here the mRNA levels of IL-2, IL-6, and IL-10 have been unaffected. The predominance of Th2-type cytokine production pattern in association with heightened HO-1 activity has been confirmed in other inflammatory conditions. Minamino and colleagues51
have demonstrated that hypoxic lungs of HO-1-overexpressing transgenic mice express significantly attenuate levels of proinflammatory cytokines like IL-1ß, IL-6, MCP-1, and macrophage inflammatory protein-2 as compared with those of wild-type animals. Sarady et al52
have studied isolated LPS-stimulated macrophages and noted that both up-regulation of HO-1 and low concentrations of exogenously administered CO significantly decrease the release of GM-CSF. In our study, LPS stimulation of both HO-1–/– and HO-1+/+ splenocytes has not resulted in increased GM-CSF levels, however, anti-CD3/anti-CD28 exposure has led to a dramatic increase in GM-CSF in the HO-1–/– compared to HO-1+/+ splenocytes.
Taken together, the aforementioned findings suggest that the HO-1 system plays a pivotal role in the early phases of immune response. Its level of activity, especially in the acute phase period, seems to determine the fate of downstream events including the profile of lymphocyte maturation (Th1 versus Th2). The utilization of the HO-1 knockout mouse model will provide a unique opportunity for evaluation and design of mechanistic studies to further our understanding of the role of HO-1 in inflammation in general and the process of transplant rejection in particular.
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Acknowledgements |
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Footnotes |
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Supported by a grant from Juvenile Diabetes Research Foundation and a National Kidney Foundation Fellowship Award (to M.H.K.).
M.H.K. and C.W. contributed equally to this work.
Accepted for publication May 25, 2004.
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 K.-J. Min, M.-s. Yang, S.-U. Ki |
